This proposal addresses several related aspects of a research problem with significant potential to influence the management of infectious diseases on a large scale, not only for vaccines against enteric bacterial pathogens, but also for vaccines against a variety of other bacteria, viruses, and parasites. The central theme of this project is for the use of attenuated mutants of Salmonella as immunologic vehicles to deliver heterologous antigens to the mucosal lymphoid tissues. This has been shown to be an effective means of stimulating significant levels of serum and mucosal antibodies as well as cell mediated immunity against the carrier and against foreign antigens delivered by these organisms. During the current funding cycle, we have addressed several broad issues relative to the use of this technique of vaccine delivery. There are, however, a number of fundamental questions remaining to be answered regarding the use of Salmonella as a vaccine carrier. When attenuated Salmonella sp. are utilized as carriers of foreign antigens, an undefined series of events occurs which can result in production of antigen specific mucosal sIgA, serum IgG, cell mediated immunity, or combinations of these immunologic events. We propose to examine a number of specific parameters that will enable us to begin to understand the mechanisms underlying these outcomes. For this competitive renewal, we propose to resolve the underlying mechanisms controlling the immunologic outcome as a function of bacterial attenuation, strain viability, bacterial species, pre-existing immunity, and qualitative and quantitative nature of the target antigen. For these experiments, animals will be inoculated orally and examined for antigen specific humoral and cellular immune responses. Isotype analysis of antigen specific antibodies from sera and mucosa will be performed by ELISA. Cellular responses will be determined by l) PCR to amplify reverse transcribed cytokine mRNAs expressed by leukocytes in vivo and 2) ELISA to determine cytokine secretion from antigen specific lymphocytes in vitro. These experiments should permit the design of specifically tailored Salmonella vaccine delivery systems according to the type and character of immune response appropriate for the target pathogen (i.e., CMI for intracellular bacteria vs. sIgA for mucosal response). In addition, we will more fully examine the issue of bacterial persistence in the tissues as a determinant of the cellular and humoral immune response and complete our studies on development of a mouse model for ETEC diarrheal disease.